Micro RNA sequencing for myocardial infarction screening

Chapter 18

Micro RNA sequencing for myocardial infarction screening Sri Harsha Kanuri1 and Rolf P. Kreutz2 1

Department of Clinical Pharmacology, Institute of Personalized Medicine (IIPM), IU School of Medicine, Indianapolis, IN, United States; 2Krannert

Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN, United States

Context Approximately 16.5 million persons in United States suffer from coronary artery disease (CAD) [1]. The lifetime risk of developing CAD in patients greater than 40 years is 32% and 49%, in men and women, respectively [2]. The risk of silent and unrecognized myocardial infarction is approximately 33% and 54%, in men and women, respectively [3]. CAD-specific mortality increase, by 29% and 48%, respectively, in men and women, is estimated to be between 1990 and 2020 in developed countries [4]. The overall death rate by myocardial infarction in the year 2015 was around 223 per 100,000 population [5]. In the United States, the healthcare costs of CAD due to hospitalizations and medications is around 108.9 billion annually [6].

Clinical profile Patient-related risk factors such as diabetes, hypertension, hyperlipidemia, smoking, and genetics can promote the process of atherosclerosis in the coronary artery intimal wall [7]. Rapid progression of atherosclerosis along with plaque rupture, plaque hemorrhage, and occlusive thrombus can lead to narrowing of coronary artery lumen, acute myocardial infarction (AMI), and sudden cardiac death (SCD) [7]. Patients typically present with ischemic chest pain, ECG abnormality, and elevated biomarkers [8]. Acute coronary syndrome with or without ST elevation myocardial infarction is usually treated with percutaneous coronary intervention (PCI), antithrombotic agents, statins, angiotensin converting enzyme inhibitors, and betablockers [8,9].

The presence of concomitant risk factors such as diabetes, hypertension, smoking, and obesity, increases the risk of developing recurrent events after initial CAD presentation [10]. Clopidogrel, a commonly used P2Y12 inhibitor, in addition to aspirin, lowered the incidence of myocardial infarction, stroke, cardiovascular deaths as compared to aspirin alone in patients with acute coronary syndrome [11]. Ticagrelor was shown to be more effective than clopidogrel, in attenuating the risk of combined endpoint of death, myocardial infarction, and stroke, in patients with acute coronary syndrome with or without ST segment elevation [12]. Prasugrel, another P2Y12 receptor antagonist, has been shown to attenuate the risk of cardiovascular death, myocardial infarction, and stroke, as compared with clopidogrel in patients with ACS treated with PCI [13]. Although dual antiplatelet therapy is effective in reducing the risk of secondary coronary events, there are limitations to the undifferentiated use of this pharmacologic treatment strategy [12]. Long-term dual antiplatelet therapy for >12 months has been shown to reduce the risk of recurrent coronary events, but at the cost of increased risk of bleeding and noncardiovascular deaths [14]. Drug-gene and drug-drug interactions of antiplatelet drugs can lead to variable treatment outcomes, in secondary prevention of recurrent coronary events [15]. In addition, recently low dose rivaroxaban was approved, for secondary prevention in patients with cardiovascular disease, in addition to low dose aspirin, further increasing antithrombotic treatment options for patients with CAD. In light of persistence of ischemic risk in some patients, and increased risk of bleeding in others, there remains a strong need for

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improved tools, at predicting subjects most likely to benefit from specific pharmacologic treatment strategies.

MiRNA as biomarkers of coronary artery disease While biomarkers of myocardial necrosis, such as cardiac troponins or creatinine-kinase-muscle brain (MB), have been used clinically in diagnosis of myocardial infarction for a long time, there currently do not exist any validated and reliable blood markers, for diagnosis of subclinical coronary artery disease. MiRNAs are increasingly being considered for early detection and treatment of stable angina, unstable angina, and myocardial infarction. Numerous studies have identified sometimes overlapping miRNA that have been associated with angiographic presence of CAD or angina pectoris.

Diagnosis of coronary artery disease Whole genome transcriptional profiling of 3 ST-elevation myocardial infarction (STEMI) patients, at baseline and 3 months follow-up, demonstrated that miR-486-3p was a potential biomarker that distinguishes patients with STEMI from stable IHD patients [16]. Analysis of 30 stable angina (SA) patients, 39 unstable angina (UA) patients, 19 AMI, and 16 healthy controls, with miRNA microarray and binary regression model demonstrated upregulation of miR-486 and MiR-92a, and their association with HDL components (HDL2, HDL3) in plasma of UA and AMI patients. The authors concluded that these miRNA may be able to differentiate between stable and unstable CAD population [17]. In an explorative analysis (TaqMan microRNA assay) of 367 miRNAs in 34 SA, 19 UA, and 20 control patients, D0 Alessandra et al. found that miR-1, mi-R122, miR-126, miR-133a, miR-133b, miR-337-5p, and miR-433 were positively modulated in UA and SA patients, and suggested as potential biomarkers in the future [18]. Measurement of miR-10 and miR-144 levels in 29 UA, 17 non-ST elevation myocardial infarction (NSTEMI), 14 STEMI, and 20 control patients demonstrated that miR-10a levels were upregulated, and miR-144 levels were downregulated in CAD patients, particularly those with STEMI and higher SYNTAX scores [19,20]. Microarray analysis and PCR validation in 5 patients with stable angina, 5 patients with STEMI, 5 patients with NSTEMI, and 5 controls, revealed that miR-941 was significantly elevated in NSTEMI or STEMI patients [21]. In a cross-sectional study consisting of 69 CAD patients and 30 control subjects, plasma samples quantified with microarray analysis and validated with RT-PCR showed that let-7c, miR-145, and miR-155 were potential biomarkers for detecting CAD in elderly patients [22].

Small RNA sequencing, RT-PCR, and bioinformatic analysis of serum from 6 UA patients, 6 STEMI, and 6 controls patients demonstrated that there are at least 38 dysregulated miRNA in UA and STEMI group. Furthermore, receiver operating characteristic (ROC) analysis revealed that miR-142-3p and miR-17-5p may be useful in diagnosis of UA and STEMI [23]. Assessment of miRNA expression by RT-PCR in 65 CAD patients, 20 UA patients, and 32 control patients of middle age (40e60 years old) demonstrated that miR-149 and miR-424 were reduced 4.49 fold and 3.6 fold, respectively, in stable CAD patients, and reduced 5.09 and 5 fold, respectively, in unstable CAD patients, as compared to non-CAD group. In contrast, miR-765 was elevated 3.98 fold and 5.33 fold, in stable and unstable CAD patients, respectively [24]. Thus, miR-149, miR424, and miR-765 were identified as potential noninvasive biomarkers for coronary artery disease patients in middle-aged patients [24]. miR-765 and miR-149 were significantly associated with coronary artery disease, in an elderly population. In 37 stable CAD patients (72.9  4.2 years), 32 unstable CAD patients (72.03  4.3 years), and 20 healthy volunteers (71.7  5.2 years) followed by real-time PCR analysis revealed that miR-765 levels were increased by 2.9 fold (stable CAD) and 5.8 fold (unstable CAD), whereas miR149 levels were decreased by 3.5 fold (stable CAD) and 4.2 fold (unstable CAD), respectively [25].

Gene polymorphisms of miRNA Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and Sequenom MassARRAY system analysis of 356 CAD patients and 368 control population showed that T-allele of rs2431697 was associated with increased, and G allele of rs2910164 of miR-146a with decreased CAD risk, in a Chinese population [26]. Metaanalysis of 1565 CAD cases and 1541 controls in PubMed, EMBASE, and Chinese National Knowledge Infrastructure (CKNI) databases showed that miR-146a gene polymorphism rs2910164 was associated with increased CAD risk, in Asians and older population [27]. Real-time PCR and restriction fragment length polymorphism (RLFP) performed in 272 CAD patients and 149 control patients demonstrated that TT genotype of miR146a SNP rs2292382 was more frequently associated with CAD risk in Iranian population [28].

Myocardial infarction TaqMan microarrays were used to measure the expression of miR-423-5p, miR-208, and miR-1 in 17 AMI, 4 SA, and 3 controls just before percutaneous coronary intervention (PCI), as well as 6, 12, and 24 h after the procedure [29].

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Nabialek et al. reported that miR-423-5p was the earliest biomarker that became upregulated with myocardial necrosis, and normalized quickly within 6 h, and thus could be useful in diagnosis of early AMI [29]. Measuring miRNA expression in an initial training cohort (35 atypical CAD and 20 controls), and a validation cohort (122 atypical CAD and 68 controls) with TaqMan Low Density Array and RT-qPCR demonstrated that upregulation of miR-208, miR-215, miR-485, and miR502, and downregulation of miR-29b was suggested as an important miRNA fingerprint that could signal the onset of atypical angina or silent myocardial infarction [30]. It was suggested that these miRNA could be studied as potential therapeutic targets, for arresting underlying pathophysiologic events such as atherosclerosis, inflammation, fibrosis, and myocardial ischemia [30]. Blood collected from 127 control patients, 176 angina pectoris patients, and 13 AMI was analyzed with ELISA and PCR, to measure miR-133a and troponin I (cTnl) levels. miR-133a levels increased by 72.1 fold within 21.6 h of onset of AMI, which closely resembles the expression pattern of cTnl [31]. miR-133a levels were associated with severity of coronary artery lesions, in CAD patients [31]. 17 STEMI patients and 7 control patients were investigated at the time of admission and 6 months following AMI, and miRNA expression profile was assessed and validated with RT-PCR. The study reported miR-22-5p as a novel and important biomarker that can be useful for diagnosis of AMI [32].

Cardiovascular risk factors and coronary artery disease In 110 CAD patients undergoing coronary angiography, real-time PCR showed that a lower level of miR-155 was associated with CAD, severity of coronary artery lesions, and proatherogenic metabolic risk factors such as hypertension, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and C-reactive protein levels [33]. Also in 30 angiographically confirmed CAD patients and 30 age matched controls, real-time PCR revealed that miR-33a level was 2.9 fold higher in CAD patients, and negatively correlated with total cholesterol, HDL, triglycerides, and VLDL [34]. Real-time PCR of blood samples from 54 patients with diabetes, 46 patients with CAD and diabetes, and 20 healthy volunteers showed that miR-126 and miR-200 were significantly upregulated in patients with CAD and diabetes, and correlated with glycemic and lipid profiles [35]. Interestingly, quantitative real-time PCR analysis of type-2 diabetes (T2D) alone, and T2D patients with CAD, showed

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that miR-126 was positively correlated with CAD in T2D, and negatively associated with LDL in CAD patients [36]. miR-9 and miR-370 were significantly upregulated in T2D and CAD & T2D patient groups [37]. This study highlights the diagnostic as well as prognostic significance of miR-9 and miR-370, in patients with T2D [37]. Isolation of monocytes from patients with smoking history and nonsmoking group, in a cohort of 76 premature CAD and healthy controls, demonstrated increased expression of miR-124-3p in smoking group [38]. Furthermore, RT-PCR of blood from smokers revealed that miR-124-3p is significantly upregulated in patients with subclinical and advanced atherosclerosis [38].

Coronary plaques and pathogenesis In 78 CAD patients and 65 controls, it was revealed that miR-206 levels were elevated in diseased endothelial progenitor cells (EPCs) and plasma of CAD patients, and suppressed angiogenesis through downregulation of vascular endothelial growth factor (VEGF) [39]. Similarly miR-23a in 13 AMI patients, 176 angina pectoris patients, and 127 control subjects with PCR revealed that miR-23a is elevated in EPCs and CAD patients, and that it attenuates epidermal growth factor receptor expression [40]. Blood and coronary artery plaques collected from CAD patients, showed that miR-365 level was decreased in plaques, and that miR-365 might be involved in mounting an immune response in CAD patients, through IL-6 production [41]. In another protocol, stepwise multivariate regression analysis demonstrated that miR-17-5p may be a useful biomarker that can reflect the severity of coronary atherosclerosis [42]. miR-21, mi-92a, and miR-99a were significantly elevated in advanced human coronary artery plaques, and implicated in various underlying pathways responsible for coronary atherosclerosis [43]. In another experience, miR-221 was elevated in coronary artery atherosclerotic plaques. It was significantly elevated in patients with risk factors such as hypertension, hypercholesterolemia, and family history of CAD [44]. Integrated backscatter intravascular ultrasound (IB-IVUS) was selected to determine the percentage of lipid volume and fibrous volume in coronary arteries [45]. Subsequently, blood collected from aorta and coronary sinus was analyzed for muscle-specific and vascular enriched miRNA [45]. miR-100 was more concentrated in the coronary sinus than in the aorta, and transcoronary gradient was positively correlated with % lipid volume, and negatively correlated with % fibrous volume of coronary plaques. Coronary artery plaque rupture results in release of miR-100 into the plasma circulation, thereby emphasizing its possible role in plaque stability in symptomatic CAD patients [45].

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Ventricular arrhythmias In a rat model of myocardial infarction and ovarian deficiency, increased risk of ventricular arrhythmias was associated with downregulation of miR-151-5p, induced upregulation of PLM (phospholemman) gene, downregulation of Kir2.1 potassium channel expression, and increased calcium overload [46]. These findings suggest that decreased expression of miR-151-5p might contribute to increased susceptibility to ventricular arrhythmias, and may have significance in biomarker discovery and development of novel therapeutic interventions against ventricular arrhythmias in CAD. Pilot microarray analysis of pooled plasma from apolipoprotein E (apoE) knockout mice showed that miR-21, miR-23a, and miR-30a were differentially expressed in apoE-mice. This was later confirmed in 32 CAD patients with angiographic stenosis greater than 70% [47].

Nitric oxide pathways In a mouse ischemic hindlimb model, injection of endothelial progenitor cells (EPCs) transfected with anti-miR206 resulted in activation of PIK3C2a, AKT, and eNOS [48]. Thus, it has been proposed that miR-206 induced downregulation of phospho-AKT/nitric oxide synthase pathway contributes to decreased angiogenesis, decreased EPC migration, and increased risk of coronary artery disease [48].

Antiangiogenic effects and other mechanisms Interestingly, miR-361-5p induced downregulation of vascular endothelial growth factor (VEGF), by binding to 30 UTR of VEGF [49]. Additionally, miR-221, miR-222, and miR-92a also exert antiangiogenic effects, thereby resulting in attenuated regenerative capacity of EPCs in CAD patients [50]. Previous reports suggest that miRNAs can modulate atherosclerosis through smooth muscle injury, endothelial damage, and plaque formation [51]. Some miRNAs can induce death of vascular endothelial cells, by promoting early senescence through expression of SIRT1 (miR-217 and miR-34), or by disorganization of cell cycle replication (miR-503), by targeting CCNE1 and CDC25A [52]. They were shown to regulate VEGF (miR361-5p) and epidermal growth factor receptor (EGFR) (miR-23a) expression, which may affect plaque stability (miR-100), vasculopathy, and severity of coronary artery stenosis (miR-17-5p) in CAD patients [40,42,45,49]. MiR-22 expression in peripheral blood mononuclear cells (PBMC) was determined in 21 SA patients, 17 NSTEMI patients, 14 STEMI patients, and 20 control patients with the help of RT-PCR. Furthermore monocyte

chemoattractant protein-1 (MCP-1) mRNA and protein levels were measured with RT-PCR and ELISA, respectively [53]. Downregulation of miR-22 in PBMC of CAD patients may participate in inflammatory response, by increasing the levels of MCP-1 protein [53].

Arterial calcification miR-32 could be involved in calcification of vascular smooth muscle through bone morphogenetic protein-1, runt related transcription factor-2 (RUNX2), osteopontin, and bone-specific phosphoprotein matrix GLA protein, in calcified mice vascular smooth muscle cells in in vitro studies [54]. miR-32 is upregulated in CAD patients with coronary artery calcification [54].

Platelets Upregulation of miR-384 and miR-624 in platelets of patients with coronary artery disease was documented, as compared to healthy controls [55]. Twenty-one NSTEMI patients receiving dual antiplatelet therapy, including newer P2Y12 antagonists, were analyzed with PCR and multiple electrode aggregometry, to measure miR-223 levels and platelet aggregation, respectively [56]. Higher miR-223 levels were correlated with more potent platelet inhibition, and better responsiveness with newer P2Y12 antagonists [56].

Prognosis of coronary artery disease miR-1, miR-133a, miR-133b, and miR-208b were associated with myocardial injury and onset of myocardial infarction. This study also reported that miR-133a and miR208b levels provide prognostic information by predicting all-cause mortality, 6 months from the onset of CAD [57]. Lowered expression of miR-145 was associated with AMI, and low circulating levels of miR-145 was predictive of impending heart failure in patients with AMI [58].

Large series In 4160 patients with AMI enrolled in Osaka Acute Coronary Insufficiency Study, 11 miRNAs were differentially expressed in serum of patients susceptible for cardiovascular death in the future. Particularly, in patients with a primary myocardial infarction event, elevated levels of miR-155 (fourfold) and miR-380 (threefold) were associated with cardiovascular death within 1 year [59]. Serum samples of 873 CAD patients revealed that upregulation of miR-197 and miR-223 might be associated with cardiovascular death, during a 4-year follow-up period, in association with acute coronary syndrome and stable angina pectoris [60,61].

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Presence of coronary collaterals In aortic blood from 41 patients undergoing coronary angiography, who were classified according to their collateral circulation, miR-423-5p, miR-10b, miR-30d, and miR-126 were significantly elevated in those with low (<0.39) collateral flow index (CFI), as compared to patients with high (>0.39) CFI [62]. Additionally, these biomarkers could also be useful for predicting patients who are at risk of low collateral capacity, which may impact the clinical outcomes of coronary artery disease patients [62].

Fingerprints for recurrent coronary events Recurrent cardiovascular events occur more frequently in patients with multiple risk factors, including diabetes mellitus [63]. In patients younger than 65 years, smoking, hypertension, and dyslipidemia are important risk factors [64]. The risk of recurrent cardiovascular events and associated mortality in patients with previously diagnosed acute coronary syndrome remains elevated despite treatment with high intensity statins [65]. N-terminal prohormone brain natriuretic peptide, cystatin C, albuminuria, C-reactive protein (CRP), lipoprotein associated phospholipaseA2, and secretory phospholipaseA2 can predict increased risk of recurrent cardiovascular events; however, sensitivity and specificity of these biomarkers remain modest [66,67]. Advantages of miRNAs for usage as biomarkers include tissue-specific expression, reproducibility, small size, stability in blood, as well as effective quantification with PCR and next generation sequencing [68]. We recently demonstrated 70 miRNA that were differentially expressed between CAD patients with recurrent events, as compared to CAD patients with no events [69]. A significant number of these 70 miRNA were associated with clinical presentation of unstable angina and myocardial infarction [69]. Interestingly, some of these differentially expressed miRNAs in CAD patients with recurrent events, were associated with underlying pathogenic mechanisms that promote coronary artery thrombosis, such as platelet activation (miR-340-3p, miR-451a, miR-1976, and miR-6734), endothelial dysfunction (miR-19b-3p, miR106-3p, miR-185-3p, and miR-589-5p), vascular smooth muscle proliferation (miR-29a-3p, miR-143-3p, miR-1523p, and miR-589-5p), angiogenesis (miR-485-3p, and miR-18a-3p), coronary artery calcification (miR-27a-3p, miR-29a-3p, miR-223, and miR-4745) and atherosclerosis (miR-10a-5p, miR-27a-3p, miR-331-3p, and miR-106b3p). Additionally, we found that three miRNAs, namely miR-6087, miR-3653, and miR-551a, were differentially expressed between subjects with recurrent events as compared with controls [69]. In coronary artery disease patients who undergo percutaneous coronary intervention,

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miR-483, miR-555, and miR-155 may serve as biomarkers that can indicate early phase of coronary artery plaque injury and rupture [70]. Downregulation of miR-199a, and upregulation of SIRT1 protein in myocardial tissue, is associated with increased risk of major adverse cardiac and cerebrovascular events (MACCEs), during 3.2 years follow-up in patients undergoing coronary artery bypass graft (CABG) [71]. In patients with coronary artery disease, upregulation of miRNA-126 is negatively associated with inflammatory markers such as C-reactive protein, tumor necrosis factoralpha (TNF-alpha), and intracellular adhesion molecule (ICAM-1), thereby resulting in decreased risk and severity of recurrent coronary events in the future [72]. miR-126 is an important biomarker that can predict the occurrence of major adverse cardiovascular events, in patients undergoing primary coronary intervention [73]. Only microvesicle associated miR-126 and miR-199, but not freely circulating miRNAs, can predict the occurrence of recurrent coronary events in stable CAD patients [74]. Upregulation of miR-133a in the coronary circulation might reflect the activation of underlying pathophysiological mechanisms leading to CAD progression and associated complications, and it can be a potential biomarker for predicting coronary artery in-stent restenosis [75]. Elevated miR-223 has been associated with increased platelet responsiveness to newer P2Y12 antagonist antiplatelet therapy, and thus might influence the occurrence of recurrent coronary events after primary CAD [56]. Upregulation of miRNA-329, miR-494, and miR-495 has been associated with increased intimal hyperplasia and atherosclerosis, thus favoring coronary artery in-stent restenosis in patients [76]. Vulnerable and unstable coronary artery plaques release miR-21, miR-100, miR-155-5p, miR-4835p, and miR451a, which can be important harbingers and predictors of impending coronary thrombosis, in patients with CAD [45,70,77]. Downregulation of miR-125a-5p, miR-155, and miR-199a/b-3p and elevation of endothelin1 in the coronary circulation can accentuate coronary atherosclerosis, and might signal the occurrence of coronary thrombosis after a primary event [77].

Current studies with miRNA in cardiovascular disease and metabolic function Increased miR-1 and decreased miR-133 lead to myocardial apoptosis, whereas decreased miR-1 and increased miR133 promote myocardial survival [78]. miR-1 regulates Hsp-60 posttranscriptionally, and leads to cardiomyocyte apoptosis [79]. A recent study reported that miR-24 affords protection against myocardial apoptosis, via inhibiting proapoptotic gene Bcl-2 [80]. miR-199 plays an important

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role in regulation of size of myocytes and cardiac hypertrophy, through hypoxia-inducible factor 1 alpha [81]. MiR-378 has been shown to offer protection against hypoxic myocardial apoptosis, through downregulation of caspase-3 expression in cardiomyocytes [82]. miR-378 overexpression can protect against doxorubicin induced myocardial apoptosis, through increased calumenin expression, and decreased endoplasmic reticulum stress response [83]. Overexpression of miR-21 has been associated with reduced myocardial apoptosis, attenuated infarct size, and improved ventricular remodeling, through programmed cell death 4 and activator 1 pathway [84]. More specifically, miR-21 induced protection against hypoxia-induced cardiomyocyte injury is mediated through inhibition of excessive autophagy and PTEN/AKT/mTOR signaling pathway [85,86]. Interestingly, the antiischemic agent trimetazine (TMZ) protected against hypoxiareperfusion induced cardiomyocyte apoptosis, by upregulating miR-21 expression [87].

miRNA inhibition In rat model of AMI, administration of anti-miR-214 resulted in attenuated apoptosis, myocardial infarct size, and improved left ventricular remodeling through PTEN (phosphatase and tense homolog) and Bim1 expression. This demonstrates the role of miR-214 on left ventricular remodeling in AMI [88,89]. Li et al. reported that, miR-23 accentuates myocardial apoptosis by increasing superoxide levels, decreasing Bax/Bcl2 protein expression ratio, caspase-3 activity, and P53 expression via PTEN dependent manner, in H9C2 cell culture model, thus endorsing its role in AMI [90]. In H9c2 cell culture model, overexpression of miR-122 results in hypoxia-induced cardiomyocyte apoptosis, whereas knockdown of miR-122 results in improved cardiomyocyte survival through PTEN/ P13K/AKT, and activation of cellular autophagy [91]. In a cell culture model utilizing human cardiac microvascular endothelial cells, miR-126, miR-130a, and miR138 afforded protection against inflammatory response and hypoxia induced injury, via P13K/AKT/eNOS signaling pathway [92e94]. Overexpression of miR-106b and miR-495 leads to protection of coronary endothelial cells from hypoxia/reperfusion induced injury, through NFkB and NLRP3 signaling mechanisms, respectively [95,96]. Transplantation of endothelial stem cells resulted in attenuation of myocardial apoptosis, via downregulation of miR-146a in a rat model of AMI [97]. Platelet-derived miR-4306 can limit the migration of human monocytee derived macrophages into cardiac tissue, in myocardial infarction mice, thereby preventing cardiac damage through left ventricular remodeling and dysfunction postinfarction [98,99].

miR-22 can lower the production of NLRP3 inflammasome induced cytokine production, and affords protection against coronary artery endothelial cells apoptosis, during hypoxic stress [100]. Downregulation of miR-499 leads to protection of coronary endothelial cells, from inflammation mediated damage, through increased PDCD4 expression and decreased NF-kB signaling pathway [101]. In vitro studies with vascular smooth muscle cells (VSMCs) demonstrate that miR-574 leads to proliferation of VSMCs and decreased apoptosis, making it a viable therapeutic target for treatment of coronary artery disease [102]. Alternatively, miR-362-3p might play a critical role, in atherosclerosis and progression of coronary artery disease, by downregulating the proliferation and migration of VSMCs via ADAMST1 [103].

Hypoxia reperfusion injury Transgenic mice overexpressing miR-125b, subjected to ischemia and reperfusion injury, demonstrated decreased myocardial infarct size, attenuated caspase 3/7/8 activity, and reduced myocardial apoptosis, via decreased TNF receptor associated factor six levels and NF-kB activation [104]. In rat H9c2 cardiomyocytes, miR-30b mediated protection against hypoxia-reperfusion induced cardiomyocyte injury via increased Bcl2, decreased Bax, downregulated caspase-3, increased AKT and decreased KRAS [105,106]. Both miR-19a and miR-93 overexpression lead to protection, of hypoxia and reoxygenation induced cardiomyocyte injury, via PTEN/P13K/pAKT pathway, in cell culture model of ischemic heart disease [107,108]. In a cell culture model utilizing neonatal rat ventricular myocytes and H9c2 cells, miR-449a protected against hypoxia induced myocyte injury, through notch-1 signaling pathway [109]. In rat cardiomyocytes subjected to hypoxic and reperfusion injury, upregulation of miR-302 leads to inhibition of antiapoptotic myeloid leukemia cell differentiation protein (Mcl-1), and activation of proapoptotic mechanisms, resulting in cardiomyocyte apoptosis [110]. Accordingly, Fang et al. reported that miR-302 antagonists can be employed as a therapeutic intervention to rescue cardiomyocytes from hypoxia-reperfusion injury induced apoptosis [110]. Downregulation of miR-320 and miR-103 can attenuate myocardial apoptosis, associated with ischemia and reperfusion injury, and thus improve myocardial function [111]. MiR-188-3p and miR-145 can attenuate hypoxia induced myocardial necrosis, by activating autophagic pathways [111]. MiR-509, miR-199a, and miR-204 can function through activation of cardiac regenerative processes and cardiomyocyte proliferation, thereby promoting cardiac repair and regeneration following myocardial infarction [111].

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Collagen and myocardial fibrosis Overexpression of miR-181a leads to deposition of extracellular matrix (collagen-1 and fibronectin) and promotion of myocardial fibrosis, following myocardial infarction in a rat model of myocardial infarction [112]. MiR-328, miR21, and miR-223 play important roles in cardiac fibrosis, by regulating deposition of fibrous tissue following myocardial infarction in animal models, via TGF-beta, Smad, RASA signaling pathways [113e115]. Interestingly, both miR-22 and miR-101a exert negative influence on cardiac fibrosis, in mouse models of myocardial infarction, by modulating TGF-beta signaling pathway [116,117].

Diagnosis and prognosis Early diagnosis of recurrent coronary events such as unstable angina or myocardial infarction, with reliable biomarkers, will be important for delivery of personalized clinical interventions in a time-dependent manner, to further improve prognosis of CAD after initial diagnosis [118]. Important features that would establish the utility of miRNAs as biomarkers are cardiac tissue specificity, rapid release kinetics, and stability in blood [57,118]. Droplet digital PCR was recently shown to have a better performance, technically and diagnostically, in quantifying miRNA levels in blood of ST-elevation MI patients, as compared to qRT-PCR in large multicenter trials [119]. miR-6090 and miR-4516 can be regarded as reference genes (optimal endogenous controls) that might be used to normalize the RT-PCR data, for quantification of miRNA expression in the plasma of CAD patients [120].

Robust diagnostic signatures miRNA-1, miRNA-133, miRNA-208b, and miRNA-499 demonstrated high sensitivity and specificity for diagnosis of acute myocardial infarction [118]. A miRNA signature consisting of miRNA-19b-3p, miR134-5p, and miRNA186-5p was upregulated within 4e72 h of onset of chest pain in acute myocardial infarction, and was also positively correlated with troponin levels [121]. miR-1, miR-133a, miR-133b, miR-208a, miR-208b, and miR-499 were elevated in patients with ST-elevation MI [57,122e124]. These six miRNAs were positively correlated with troponin (hSTnT), and negatively associated with left ventricular ejection fraction [57,125]. However, previous studies indicate that these cardio-specific miRNAs, are not superior to high sensitivity troponin, which is routinely used for diagnosis of early stage of acute myocardial infarction [126]. miR-1, miR-133a, and miR208a are elevated as early as 120 min, and return to baseline within 12 h following an episode of acute myocardial infarction [125]. In contrast, miR-499-5p

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becomes elevated within 150 min, and returns to baseline with 2e3 days after myocardial infarction [125]. Interestingly, miR-1, miR-133a, and miR-208b were more elevated in patients with myocardial infarction (STEMI or NSTEMI), as compared to patients with unstable angina [31,57]. A miRNA signature consisting of miR196-5p, miR-3163-3p, miR-143-3p, and miR-190a-5p may be useful as novel diagnostic markers for diagnosis of very early onset CAD, in patients with ages around 40 years [127]. miR-21-5p, miR-19b-3p, miR-30d-5p, miR-122-5p, miR-125b-5p, miR134-5p, miR-146a, miR-186-5p, miR221-3p, miR-320a, miR-328, miR361-5p, miR-375, miR423, and miR-519e-5p can also be considered as potential and promising new biomarkers for early diagnosis of acute myocardial infarction [121,128e132]. Following an AMI, miR-133, miR-208b, and miR-499b can also provide prognostic significance, by predicting heart failure and allcause mortality within 6 months [57,122]. Overexpression of miR-23a leads to attenuated expression of telomeric repeat binding factor (TRF2), lower telomere length, and accelerated coronary atherosclerosis, which is associated with poor clinical prognosis [133]. The genotype CC/CT of hsa-miR-196a2 rs11614913, along with diabetes, smoking, age, and pathological changes in coronary arteries was associated with severe prognosis in CAD in Chinese patients [134]. miR-155 and miR-503 are associated with formation of coronary collateral circulation, and thus may affect prognosis in coronary artery disease patients [135,136]. miRNA-related polymorphisms such as miR-4513 (rs2168518) and miR-499 (rs3746444) might be potential biomarkers that can indicate clinical prognosis of CAD patients [137]. miR-126 is positively associated with coronary collateral circulation, and its upregulation may be used to predict collateral formation in ischemic myocardium, supplied by severely stenosed coronary arteries [138]. According to Wang et al., endothelial cell associated miRNAs, such as miR-31 and miR-720, have the potential to be used as reliable biomarkers, for both earlier diagnosis and clinical prognosis of CAD [139].

Challenges and pitfalls Factors that can influence plasma miRNA levels encompass heparin, statins, ACE inhibitors, antiplatelet therapy, kidney disease, cancer, and cytomegalovirus infection [140]. Additionally, other disadvantages of miRNA that need consideration include the absence of reference threshold values, as well as standardized methods of RNA preparation and normalization methods [140]. Next generation sequencing, qPCR, and microarrays are most commonly used [140]. These methods are very time-consuming, and cannot be used for bedside diagnosis of unstable angina and

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STEMI [140]. In the future, programmable oligonucleotide probes that can detect single molecules of miRNA in plasma may need to be developed [140].

Future research There are around 20 miRNAs that can be considered as potential biomarkers for diagnosis of acute myocardial infarction, yet most of these were demonstrated in small study cohorts with small sample sizes, leading to wide variance in findings. Larger study cohorts with validation are advised [141]. Further research is particularly warranted in areas such as origin, regulation, function, and end-targets of candidate miRNAs [142]. Currently, it takes around 2e3 h for quantification of specific miRNA in blood with PCR [143]. Automated work flow systems that can rapidly, specifically, and sensitively detect very low levels of miRNA in plasma at the bedside should be developed [142,143]. Furthermore, research is needed to examine how drugs (e.g., heparin, statin, ACE inhibitors) and other clinical variables may influence miRNA levels in plasma [142]. The role of endothelial, platelet, coronary, and vascular smooth muscle cellespecific miRNA, and the role they play in the pathogenesis of coronary artery disease require further characterization. miRNA role in pathophysiology of CAD disease progression and complications using in vitro and animal studies are necessary [144]. Biomaterial standardization (serum, plasma, whole blood), and RNA normalization procedures for accurate quantification of miRNA levels are relevant expectations [144]. Lastly, large clinical studies, enrolling subjects from multiple racial and ethnic groups, utilizing standardized procedures for RNA extraction, will need to confirm the utility of candidate miRNA biomarkers for detecting stable angina, unstable angina, and acute myocardial infarction [145].

References [1] Benjamin EJ, Blaha MJ, Chiuve SE, et al. Heart disease and stroke statistics-2017 update: a report from the American heart association. Circulation 2017;135(10):e146e603. [2] Lerner DJ, Kannel WB. Patterns of coronary heart disease morbidity and mortality in the sexes: a 26-year follow-up of the Framingham population. Am. Heart J. 1986;111(2):383e90. [3] de Torbal A, Boersma E, Kors JA, et al. Incidence of recognized and unrecognized myocardial infarction in men and women aged 55 and older: the Rotterdam study. Eur. Heart J. 2006;27(6):729e36. [4] Sanchis-Gomar F, Perez-Quilis C, Leischik R, Lucia A. Epidemiology of coronary heart disease and acute coronary syndrome. Ann. Transl. Med. 2016;4(13):256. [5] Benjamin EJ, Virani SS, Callaway CW, et al. Heart disease and stroke statistics-2018 update: a report from the American heart association. Circulation 2018;137(12):e67e492.

[6] Million hearts: strategies to reduce the prevalence of leading cardiovascular disease risk factorsdUnited States, 2011. MMWR Morb. Mortal. Wkly. Rep. 2011;60(36):1248e51. [7] Ambrose J, Singh M. Pathophysiology of coronary artery disease leading to acute coronary syndromes. 2015. [8] Fox KAA. Acute coronary syndromes: presentationdclinical spectrum and management. Heart 2000;84(1):93. [9] API expert consensus document on management of ischemic heart disease. J. Assoc. Physicians India 2006;54:469e80. [10] Hajar R. Risk factors for coronary artery disease: historical perspectives. Heart Views 2017;18(3):109e14. [11] Knight CJ. Antiplatelet treatment in stable coronary artery disease. Heart 2003;89(10):1273e8. [12] Wallentin L, Becker RC, Budaj A, et al. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N. Engl. J. Med. 2009;361(11):1045e57. [13] Smith JN, Negrelli JM, Manek MB, Hawes EM, Viera AJ. Diagnosis and management of acute coronary syndrome: an evidencebased update. J. Am. Board Fam. Med. 2015;28(2):283e93. [14] Wilson SJ, Newby DE, Dawson D, Irving J, Berry C. Duration of dual antiplatelet therapy in acute coronary syndrome. Heart 2017;103(8):573. [15] Eltayeb H, Nelson A, Willoughby S, Worthley M. Antiplatelet therapy in acute coronary syndromes: current agents and impact on patient outcomes. 2011. [16] Wei T, Folkersen L, Ehrenborg E, Gabrielsen A. MicroRNA 4863P as a stability marker in acute coronary syndrome. Biosci. Rep. 2016;36(3). [17] Niculescu LS, Simionescu N, Sanda GM, et al. MiR-486 and miR92a identified in circulating HDL discriminate between stable and vulnerable coronary artery disease patients. PLoS One 2015;10(10):e0140958. [18] D’Alessandra Y, Carena MC, Spazzafumo L, et al. Diagnostic potential of plasmatic MicroRNA signatures in stable and unstable angina. PLoS One 2013;8(11):e80345. [19] Chen B, Luo L, Wei X, Gong D, Jin L. Altered plasma miR-144 as a novel biomarker for coronary artery disease. Ann. Clin. Lab. Sci. 2018;48(4):440e5. [20] Luo L, Chen B, Li S, et al. Plasma miR-10a: a potential biomarker for coronary artery disease. Dis. Mark. 2016;2016:3841927. [21] Bai R, Yang Q, Xi R, Li L, Shi D, Chen K. miR-941 as a promising biomarker for acute coronary syndrome. BMC Cardiovasc. Disord. 2017;17(1):227. [22] Faccini J, Ruidavets J-B, Cordelier P, et al. Circulating miR-155, miR-145 and let-7c as diagnostic biomarkers of the coronary artery disease. Sci. Rep. 2017;7:42916. [23] Zhong Z, Hou J, Zhang Q, et al. Circulating microRNA expression profiling and bioinformatics analysis of dysregulated microRNAs of patients with coronary artery disease. Medicine 2018;97(27):e11428. [24] Sayed AS, Xia K, Li F, et al. The diagnostic value of circulating microRNAs for middle-aged (40-60-year-old) coronary artery disease patients. Clinics 2015;70(4):257e63. [25] Ali Sheikh MS, Xia K, Li F, et al. Circulating miR-765 and miR149: potential noninvasive diagnostic biomarkers for geriatric coronary artery disease patients. BioMed Res. Int. 2015;2015: 740301.

Micro RNA sequencing for myocardial infarction screening Chapter | 18

[26] Wang Y, Wang X, Li Z, et al. Two single nucleotide polymorphisms (rs2431697 and rs2910164) of miR-146a are associated with risk of coronary artery disease. Int. J. Environ. Res. Public Health 2017;14(5). [27] Zhou HY, Wei Q, Shi XD, Cao HY, Qin L. miR-146a rs2910164 polymorphism might be associated with coronary artery disease risk in Asians. Cell. Mol. Biol. 2017;63(8):27e9. [28] Ghaffarzadeh M, Ghaedi H, Alipoor B, et al. Association of MiR149 (RS2292832) variant with the risk of coronary artery disease. J. Med. Biochem. 2017;36(3):251e8. [29] Nabialek E, Wanha W, Kula D, et al. Circulating microRNAs (miR423-5p, miR-208a and miR-1) in acute myocardial infarction and stable coronary heart disease. Minerva Cardioangiol. 2013;61(6):627e37. [30] Wang J, Pei Y, Zhong Y, Jiang S, Shao J, Gong J. Altered serum microRNAs as novel diagnostic biomarkers for atypical coronary artery disease. PLoS One 2014;9(9):e107012. [31] Wang F, Long G, Zhao C, et al. Plasma microRNA-133a is a new marker for both acute myocardial infarction and underlying coronary artery stenosis. J. Transl. Med. 2013;11:222. [32] Maciejak A, Kiliszek M, Opolski G, et al. miR-22-5p revealed as a potential biomarker involved in the acute phase of myocardial infarction via profiling of circulating microRNAs. Mol. Med. Rep. 2016;14(3):2867e75. [33] Zhu GF, Yang LX, Guo RW, et al. microRNA-155 is inversely associated with severity of coronary stenotic lesions calculated by the Gensini score. Coron. Artery Dis. 2014;25(4):304e10. [34] Reddy LL, Shah SA, Ponde CK, Rajani RM, Ashavaid TF. Circulating miRNA-33: a potential biomarker in patients with Coronary Artery Disease (CAD). Biomarkers: biochemical indicators of exposure, response, and susceptibility to chemicals. 2018. p. 1e27. [35] Amr KS, Abdelmawgoud H, Ali ZY, Shehata S, Raslan HM. Potential value of circulating microRNA-126 and microRNA-210 as biomarkers for type 2 diabetes with coronary artery disease. Br. J. Biomed. Sci. 2018;75(2):82e7. [36] Al-Kafaji G, Al-Mahroos G, Abdulla Al-Muhtaresh H, Sabry MA, Abdul Razzak R, Salem AH. Circulating endothelium-enriched microRNA-126 as a potential biomarker for coronary artery disease in type 2 diabetes mellitus patients. Biomarkers 2017;22(3e4):268e78. [37] Motawae TM, Ismail MF, Shabayek MI, Seleem MM. MicroRNAs 9 and 370 association with biochemical markers in T2D and CAD complication of T2D. PLoS One 2015;10(5):e0126957. [38] de Ronde MWJ, Kok MGM, Moerland PD, et al. High miR-124-3p expression identifies smoking individuals susceptible to atherosclerosis. Atherosclerosis 2017;263:377e84. [39] Wang M, Ji Y, Cai S, Ding W. MiR-206 suppresses the progression of coronary artery disease by modulating vascular endothelial growth factor (VEGF) expression. Med. Sci. Monit. 2016;22:5011e20. [40] Wang S, He W, Wang C. MiR-23a regulates the vasculogenesis of coronary artery disease by targeting epidermal growth factor receptor. Cardiovasc. Ther. 2016;34(4):199e208. [41] Lin B, Feng DG, Wang F, et al. MiR-365 participates in coronary atherosclerosis through regulating IL-6. Eur. Rev. Med. Pharmacol. Sci. 2016;20(24):5186e92.

195

[42] Chen J, Xu L, Hu Q, Yang S, Zhang B, Jiang H. MiR-17-5p as circulating biomarkers for the severity of coronary atherosclerosis in coronary artery disease. Int. J. Cardiol. 2015;197:123e4. [43] Parahuleva MS, Lipps C, Parviz B, et al. MicroRNA expression profile of human advanced coronary atherosclerotic plaques. Sci. Rep. 2018;8(1):7823. [44] Bildirici AE, Arslan S, Ozbilum Sahin N, Berkan O, Beton O, Yilmaz MB. MicroRNA-221/222 expression in atherosclerotic coronary artery plaque versus internal mammarian artery and in peripheral blood samples. Biomarkers 2018;23(7):670e5. [45] Soeki T, Yamaguchi K, Niki T, et al. Plasma microRNA-100 is associated with coronary plaque vulnerability. Circ. J. 2015;79(2):413e8. [46] Zhang Y, Wang R, Du W, et al. Downregulation of miR-151-5p contributes to increased susceptibility to arrhythmogenesis during myocardial infarction with estrogen deprivation. PLoS One 2013;8(9):e72985. [47] Han H, Qu G, Han C, et al. MiR-34a, miR-21 and miR-23a as potential biomarkers for coronary artery disease: a pilot microarray study and confirmation in a 32 patient cohort. Exp. Mol. Med. 2015;47:e138. [48] Tang Y, Zhang Y, Chen Y, Xiang Y, Xie Y. Role of the microRNA, miR-206, and its target PIK3C2alpha in endothelial progenitor cell function - potential link with coronary artery disease. FEBS J. 2015;282(19):3758e72. [49] Wang HW, Lo HH, Chiu YL, et al. Dysregulated miR-361-5p/ VEGF axis in the plasma and endothelial progenitor cells of patients with coronary artery disease. PLoS One 2014;9(5):e98070. [50] Zhang Q, Kandic I, Kutryk MJ. Dysregulation of angiogenesisrelated microRNAs in endothelial progenitor cells from patients with coronary artery disease. Biochem. Biophys. Res. Commun. 2011;405(1):42e6. [51] Wang R, Dong LD, Meng XB, Shi Q, Sun WY. Unique microRNA signatures associated with early coronary atherosclerotic plaques. Biochem. Biophys. Res. Commun. 2015;464(2):574e9. [52] Caporali A, Emanueli C. MicroRNA regulation in angiogenesis. Vasc. Pharmacol. 2011;55(4):79e86. [53] Chen B, Luo L, Zhu W, et al. miR-22 contributes to the pathogenesis of patients with coronary artery disease by targeting MCP1: an observational study. Medicine 2016;95(33):e4418. [54] Liu J, Xiao X, Shen Y, et al. MicroRNA-32 promotes calcification in vascular smooth muscle cells: implications as a novel marker for coronary artery calcification. PLoS One 2017;12(3): e0174138. [55] Sondermeijer BM, Bakker A, Halliani A, et al. Platelets in patients with premature coronary artery disease exhibit upregulation of miRNA340* and miRNA624*. PLoS One 2011;6(10):e25946. [56] Chyrchel B, Toton-Zuranska J, Kruszelnicka O, et al. Association of plasma miR-223 and platelet reactivity in patients with coronary artery disease on dual antiplatelet therapy: a preliminary report. Platelets 2015;26(6):593e7. [57] Widera C, Gupta SK, Lorenzen JM, et al. Diagnostic and prognostic impact of six circulating microRNAs in acute coronary syndrome. J. Mol. Cell. Cardiol. 2011;51(5):872e5. [58] Zhang M, Cheng YJ, Sara JD, et al. Circulating MicroRNA-145 is associated with acute myocardial infarction and heart failure. Chin. Med. J. 2017;130(1):51e6.

196 PART | II Precision medicine for practitioners

[59] Matsumoto S, Sakata Y, Nakatani D, et al. A subset of circulating microRNAs are predictive for cardiac death after discharge for acute myocardial infarction. Biochem. Biophys. Res. Commun. 2012;427(2):280e4. [60] Orenes-Piñero E, Marín F, Lip GYH. miRNA-197 and miRNA-223 and cardiovascular death in coronary artery disease patients. Ann. Transl. Med. 2016;4(10):200. [61] Schulte C, Molz S, Appelbaum S, et al. miRNA-197 and miRNA223 predict cardiovascular death in a cohort of patients with symptomatic coronary artery disease. PLoS One 2016;10(12):e0145930. [62] Hakimzadeh N, Nossent AY, van der Laan AM, et al. Circulating MicroRNAs characterizing patients with insufficient coronary collateral artery function. PLoS One 2015;10(9):e0137035. [63] van der Heijden AAWA, Van’t Riet E, Bot SDM, et al. Risk of a recurrent cardiovascular event in individuals with type 2 diabetes or intermediate hyperglycemia: the Hoorn study. Diabetes Care 2013;36(11):3498e502. [64] van den Berg MJ, Westerink J, van der Graaf Y, et al. Risk factors for recurrent cardiovascular events before age 65 years or within 2.5 years of a recent first cardiovascular event. Am. J. Cardiol. 2017;120(2):167e73. [65] Rockberg J, Jorgensen L, Taylor B, Sobocki P, Johansson G. Risk of mortality and recurrent cardiovascular events in patients with acute coronary syndromes on high intensity statin treatment. Prev. Med. Rep. 2017;6:203e9. [66] Shlipak MG, Ix JH, Bibbins-Domingo K, Lin F, Whooley MA. Biomarkers to predict recurrent cardiovascular disease: the Heart and Soul Study. Am. J. Med. 2008;121(1):50e7. [67] Wang J, Tan G-J, Han L-N, Bai Y-Y, He M, Liu H-B. Novel biomarkers for cardiovascular risk prediction. J. Geriatr. Cardiol. 2017;14(2):135e50. [68] Kaudewitz D, Zampetaki A, Mayr M. MicroRNA biomarkers for coronary artery disease? Curr. Atheroscler. Rep. 2015;17(12):70. [69] Kanuri SH, Ipe J, Kassab K, et al. Next generation MicroRNA sequencing to identify coronary artery disease patients at risk of recurrent myocardial infarction. Atherosclerosis 2018;278:232e9. [70] Li S, Lee C, Song J, et al. Circulating microRNAs as potential biomarkers for coronary plaque rupture. Oncotarget 2017;8(29):48145e56. [71] Yamac AH, Huyut MA, Yilmaz E, et al. MicroRNA 199a is downregulated in patients after coronary artery bypass graft surgery and is associated with increased levels of sirtuin 1 (SIRT 1) protein and major adverse cardiovascular events at 3-year follow-up. Med. Sci. Monit. 2018;24:6245e54. [72] Wu H, Zhang J. miR-126 in peripheral blood mononuclear cells negatively correlates with risk and severity and is associated with inflammatory cytokines as well as intercellular adhesion molecule-1 in patients with coronary artery disease. Cardiology 2018;139(2):110e8. [73] Yu XY, Chen JY, Zheng ZW, et al. Plasma miR-126 as a potential marker predicting major adverse cardiac events in dual antiplatelettreated patients after percutaneous coronary intervention. EuroIntervention 2013;9(5):546e54. [74] Jansen F, Yang X, Proebsting S, et al. MicroRNA expression in circulating microvesicles predicts cardiovascular events in patients with coronary artery disease. J. Am. Heart Assoc. 2014;3(6):e001249.

[75] De Rosa R, De Rosa S, Leistner D, et al. Transcoronary concentration gradient of microRNA-133a and outcome in patients with coronary artery disease. Am. J. Cardiol. 2017;120(1):15e24. [76] Welten SMJ, de Jong RCM, Wezel A, et al. Inhibition of 14q32 microRNA miR-495 reduces lesion formation, intimal hyperplasia and plasma cholesterol levels in experimental restenosis. Atherosclerosis 2017;261:26e36. [77] Fan X, Wang E, Wang X, Cong X, Chen X. MicroRNA-21 is a unique signature associated with coronary plaque instability in humans by regulating matrix metalloproteinase-9 via reversioninducing cysteine-rich protein with Kazal motifs. Exp. Mol. Pathol. 2014;96(2):242e9. [78] Xu C, Lu Y, Pan Z, et al. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes. J. Cell Sci. 2007;120(Pt 17):3045e52. [79] Shan ZX, Lin QX, Deng CY, et al. miR-1/miR-206 regulate Hsp60 expression contributing to glucose-mediated apoptosis in cardiomyocytes. FEBS Lett. 2010;584(16):3592e600. [80] Li DF, Tian J, Guo X, et al. Induction of microRNA-24 by HIF-1 protects against ischemic injury in rat cardiomyocytes. Physiol. Res. 2012;61(6):555e65. [81] Song XW, Li Q, Lin L, et al. MicroRNAs are dynamically regulated in hypertrophic hearts, and miR-199a is essential for the maintenance of cell size in cardiomyocytes. J. Cell. Physiol. 2010;225(2):437e43. [82] Fang J, Song XW, Tian J, et al. Overexpression of microRNA-378 attenuates ischemia-induced apoptosis by inhibiting caspase-3 expression in cardiac myocytes. Apoptosis 2012;17(4):410e23. [83] Wang Y, Cui X, Wang Y, et al. Protective effect of miR378* on doxorubicin-induced cardiomyocyte injury via calumenin. J. Cell. Physiol. 2018;233(10):6344e51. [84] Dong S, Cheng Y, Yang J, et al. MicroRNA expression signature and the role of microRNA-21 in the early phase of acute myocardial infarction. J. Biol. Chem. 2009;284(43):29514e25. [85] Huang Z, Wu S, Kong F, et al. MicroRNA-21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway. J. Cell Mol. Med. 2017;21(3):467e74. [86] Yang Q, Yang K, Li A. microRNA-21 protects against ischemiareperfusion and hypoxia-reperfusion-induced cardiocyte apoptosis via the phosphatase and tensin homolog/Akt-dependent mechanism. Mol. Med. Rep. 2014;9(6):2213e20. [87] Yang Q, Yang K, Li AY. Trimetazidine protects against hypoxiareperfusion-induced cardiomyocyte apoptosis by increasing microRNA-21 expression. Int. J. Clin. Exp. Pathol. 2015;8(4):3735e41. [88] Yang X, Qin Y, Shao S, et al. MicroRNA-214 inhibits left ventricular remodeling in an acute myocardial infarction rat model by suppressing cellular apoptosis via the phosphatase and tensin homolog (PTEN). Int. Heart J. 2016;57(2):247e50. [89] Wang X, Ha T, Hu Y, et al. MicroRNA-214 protects against hypoxia/reoxygenation induced cell damage and myocardial ischemia/reperfusion injury via suppression of PTEN and Bim1 expression. Oncotarget 2016;7(52):86926e36. [90] Li S, Ren J, Sun Q. The expression of microRNA-23a regulates acute myocardial infarction in patients and in vitro through targeting PTEN. Mol. Med. Rep. 2018;17(5):6866e72.

Micro RNA sequencing for myocardial infarction screening Chapter | 18

[91] Zhang Z, Li H, Chen S, Li Y, Cui Z, Ma J. Knockdown of MicroRNA-122 protects H9c2 cardiomyocytes from hypoxiainduced apoptosis and promotes autophagy. Med. Sci. Monit. 2017;23:4284e90. [92] Yang HH, Chen Y, Gao CY, Cui ZT, Yao JM. Protective effects of MicroRNA-126 on human cardiac microvascular endothelial cells against hypoxia/reoxygenation-induced injury and inflammatory response by activating PI3K/Akt/eNOS signaling pathway. Cell. Physiol. Biochem. 2017;42(2):506e18. [93] Song CL, Liu B, Shi YF, et al. MicroRNA-130a alleviates human coronary artery endothelial cell injury and inflammatory responses by targeting PTEN via activating PI3K/Akt/eNOS signaling pathway. Oncotarget 2016;7(44):71922e36. [94] Li JB, Wang HY, Yao Y, et al. Overexpression of microRNA-138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway. J. Cell Mol. Med. 2017;21(8):1482e91. [95] An Z, Yang G, Nie W, Ren J, Wang D. MicroRNA-106b overexpression alleviates inflammation injury of cardiac endothelial cells by targeting BLNK via the NF-kappaB signaling pathway. J. Cell. Biochem. 2018;119(4):3451e63. [96] Zhou T, Xiang DK, Li SN, Yang LH, Gao LF, Feng C. MicroRNA495 ameliorates cardiac microvascular endothelial cell injury and inflammatory reaction by suppressing the NLRP3 inflammasome signaling pathway. Cell. Physiol. Biochem. 2018;49(2):798e815. [97] Fang Y, Chen S, Liu Z, et al. Endothelial stem cells attenuate cardiac apoptosis via downregulating cardiac microRNA-146a in a rat model of coronary heart disease. Exp. Ther. Med 2018;16(5):4246e52. [98] Yang Y, Luo H, Liu S, et al. Platelet microparticles-containing miR-4306 inhibits human monocyte-derived macrophages migration through VEGFA/ERK1/2/NF-kappaB signaling pathways. Clin. Exp. Hypertens. 2018:1e11. [99] White DA, Su Y, Kanellakis P, et al. Differential roles of cardiac and leukocyte derived macrophage migration inhibitory factor in inflammatory responses and cardiac remodelling post myocardial infarction. J. Mol. Cell. Cardiol. 2014;69:32e42. [100] Huang WQ, Wei P, Lin RQ, Huang F. Protective effects of microrna-22 against endothelial cell injury by targeting NLRP3 through suppression of the inflammasome signaling pathway in a rat model of coronary heart disease. Cell. Physiol. Biochem. 2017;43(4):1346e58. [101] Zhang YH, He K, Shi G. Effects of MicroRNA-499 on the inflammatory damage of endothelial cells during coronary artery disease via the targeting of PDCD4 through the NF-Kappabeta/ TNF-alpha signaling pathway. Cell. Physiol. Biochem. 2017;44(1):110e24. [102] Lai Z, Lin P, Weng X, et al. MicroRNA-574-5p promotes cell growth of vascular smooth muscle cells in the progression of coronary artery disease. Biomed. Pharmacother. 2018;97:162e7. [103] Li M, Liu Q, Lei J, Wang X, Chen X, Ding Y. MiR-362-3p inhibits the proliferation and migration of vascular smooth muscle cells in atherosclerosis by targeting ADAMTS1. Biochem. Biophys. Res. Commun. 2017;493(1):270e6. [104] Wang X, Ha T, Zou J, et al. MicroRNA-125b protects against myocardial ischaemia/reperfusion injury via targeting p53-mediated apoptotic signalling and TRAF6. Cardiovasc. Res. 2014;102(3):385e95.

197

[105] Li T, Sun ZL, Xie QY. Protective effect of microRNA-30b on hypoxia/reoxygenation-induced apoptosis in H9C2 cardiomyocytes. Gene 2015;561(2):268e75. [106] Song CL, Liu B, Wang JP, et al. Anti-apoptotic effect of microRNA-30b in early phase of rat myocardial ischemiareperfusion injury model. J. Cell. Biochem. 2015;116(11):2610e9. [107] Sun G, Lu Y, Li Y, et al. miR-19a protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via PTEN/PI3K/p-Akt pathway. Biosci. Rep. 2017;37(6). [108] Ke ZP, Xu P, Shi Y, Gao AM. MicroRNA-93 inhibits ischemiareperfusion induced cardiomyocyte apoptosis by targeting PTEN. Oncotarget 2016;7(20):28796e805. [109] Cheng J, Wu Q, Lv R, et al. MicroRNA-449a inhibition protects H9C2 cells against hypoxia/reoxygenation-induced injury by targeting the notch-1 signaling pathway. Cell. Physiol. Biochem. 2018;46(6):2587e600. [110] Fang YC, Yeh CH. Inhibition of miR-302 suppresses hypoxiareoxygenation-induced H9c2 cardiomyocyte death by regulating Mcl-1 expression. Oxid. Med. Cell. Longev. 2017;2017: 7968905. [111] Sun T, Dong Y-H, Du W, et al. The role of MicroRNAs in myocardial infarction: from molecular mechanism to clinical application. Int. J. Mol. Sci. 2017;18(4). [112] Chen P, Pan J, Zhang X, Shi Z, Yang X. The role of MicroRNA181a in myocardial fibrosis following myocardial infarction in a rat model. Med. Sci. Monit. 2018;24:4121e7. [113] Zhao D, Li C, Yan H, et al. Cardiomyocyte derived miR-328 promotes cardiac fibrosis by paracrinely regulating adjacent fibroblasts. Cell. Physiol. Biochem. 2018;46(4):1555e65. [114] Yuan J, Chen H, Ge D, et al. Mir-21 promotes cardiac fibrosis after myocardial infarction via targeting Smad7. Cell. Physiol. Biochem. 2017;42(6):2207e19. [115] Liu X, Xu Y, Deng Y, Li H. MicroRNA-223 regulates cardiac fibrosis after myocardial infarction by targeting RASA1. Cell. Physiol. Biochem. 2018;46(4):1439e54. [116] Hong Y, Cao H, Wang Q, et al. MiR-22 may suppress fibrogenesis by targeting TGFbetaR I in cardiac fibroblasts. Cell. Physiol. Biochem. 2016;40(6):1345e53. [117] Zhao X, Wang K, Liao Y, et al. MicroRNA-101a inhibits cardiac fibrosis induced by hypoxia via targeting TGFbetaRI on cardiac fibroblasts. Cell. Physiol. Biochem. 2015;35(1):213e26. [118] Kränkel N, Blankenberg S, Zeller T. Early detection of myocardial infarction-microRNAs right at the time? Ann. Transl. Med. 2016;4(24):502. [119] Robinson S, Follo M, Haenel D, et al. Droplet digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction. Int. J. Cardiol. 2018;257:247e54. [120] Zhang Y, Tang W, Peng L, Tang J, Yuan Z. Identification and validation of microRNAs as endogenous controls for quantitative polymerase chain reaction in plasma for stable coronary artery disease. Cardiol. J. 2016;23(6):694e703. [121] Wang KJ, Zhao X, Liu YZ, et al. Circulating MiR-19b-3p, MiR134-5p and MiR-186-5p are promising novel biomarkers for early diagnosis of acute myocardial infarction. Cell. Physiol. Biochem. 2016;38(3):1015e29. [122] Gidlof O, Smith JG, Miyazu K, et al. Circulating cardio-enriched microRNAs are associated with long-term prognosis following myocardial infarction. BMC Cardiovasc. Disord. 2013;13:12.

198 PART | II Precision medicine for practitioners

[123] Xiao J, Shen B, Li J, et al. Serum microRNA-499 and microRNA208a as biomarkers of acute myocardial infarction. Int. J. Clin. Exp. Med. 2014;7(1):136e41. [124] Wang Q, Ma J, Jiang Z, Wu F, Ping J, Ming L. Identification of microRNAs as diagnostic biomarkers for acute myocardial infarction in Asian populations: a systematic review and meta-analysis. Medicine 2017;96(24):e7173. [125] Gidlof O, Andersson P, van der Pals J, Gotberg M, Erlinge D. Cardiospecific microRNA plasma levels correlate with troponin and cardiac function in patients with ST elevation myocardial infarction, are selectively dependent on renal elimination, and can be detected in urine samples. Cardiology 2011;118(4):217e26. [126] Li YQ, Zhang MF, Wen HY, et al. Comparing the diagnostic values of circulating microRNAs and cardiac troponin T in patients with acute myocardial infarction. Clinics (Sao Paulo, Brazil) 2013;68(1):75e80. [127] Du Y, Yang SH, Li S, et al. Circulating MicroRNAs as novel diagnostic biomarkers for very early-onset (
[134] Zhi H, Wang L, Ma G, et al. Polymorphisms of miRNAs genes are associated with the risk and prognosis of coronary artery disease. Clin. Res. Cardiol. 2012;101(4):289e96. [135] Fei Y, Hou J, Xuan W, Zhang C, Meng X. The relationship of plasma miR-503 and coronary collateral circulation in patients with coronary artery disease. Life Sci. 2018;207:145e51. [136] Wang J, Yan Y, Song D, Liu L, Liu B. The association of plasma miR-155 and VCAM-1 levels with coronary collateral circulation. Biomark. Med. 2017;11(2):125e31. [137] Li Q, Chen L, Chen D, Wu X, Chen M. Influence of microRNArelated polymorphisms on clinical outcomes in coronary artery disease. Am. J. Tourism Res. 2015;7(2):393e400. [138] Nie X, Su L, Zhou Y, et al. [Association between plasma levels of microRNA-126 and coronary collaterals in patients with coronary artery disease]. Zhonghua Xin Xue Guan Bing Za Zhi 2014;42(7):561e5. [139] Wang HW, Huang TS, Lo HH, et al. Deficiency of the microRNA31-microRNA-720 pathway in the plasma and endothelial progenitor cells from patients with coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 2014;34(4):857e69. [140] Paiva S, Agbulut O. MiRroring the multiple potentials of MicroRNAs in acute myocardial infarction. Front. Cardiovasc. Med. 2017;4(73). [141] Sayed ASM, Xia K, Yang T-L, Peng J. Circulating microRNAs: a potential role in diagnosis and prognosis of acute myocardial infarction. Dis. Mark. 2013;35(5):561e6. [142] Paiva S, Agbulut O. MiRroring the multiple potentials of MicroRNAs in acute myocardial infarction. Front. Cardiovasc. Med. 2017;4:73. [143] Li C, Pei F, Zhu X, Duan DD, Zeng C. Circulating microRNAs as novel and sensitive biomarkers of acute myocardial Infarction. Clin. Biochem. 2012;45(10e11):727e32. [144] Schulte C, Zeller T. microRNA-based diagnostics and therapy in cardiovascular disease-Summing up the facts. Cardiovasc. Diagn. Ther. 2015;5(1):17e36. [145] Contu R, Latronico MV, Condorelli G. Circulating microRNAs as potential biomarkers of coronary artery disease: a promise to be fulfilled? Circ. Res. 2010;107(5):573e4.